Adenylate cyclase (ATP pyrophosphate-lyase [cycling], AC, EC 4.6.1.1.), the catalytic protein that converts ATP to adenosine 3',5'-cyclic monophosphate (cAMP), plays a critical role in the signal transduction cascade of a number of fundamental hormones and neurotransmitters. For example, measurement of adenylate cyclase activity has been employed to study the altered physiology exhibited by transplanted human hearts and in congestive heart failure. See K. G. Lurie et at., J. Thorac. Cardiovasc. Surg., 86, 195 (1983); M. R. Bristow et at., New Engl. J. Med., 307, 205 (1982). (Chemical names of abbreviated compounds are given in the "Table of Abbreviations" hereinbelow).
However, a more clear elucidation of the biological role of adenylate cyclase in these and other conditions has been limited by the difficulty in monitoring accurately changes in the tissue levels of cAMP, the cyclic nucleotide that results from the reaction catalyzed by adenylate cyclase. The analytical difficulties arise because of the extremely low concentrations of 3',5'-cAMP in most mammalian tissues and the similarity of this cyclic nucleotide to other naturally occurring, potentially interfering, nucleotides that are present in several hundred to several hundred thousand times the concentration of 3',5'-cAMP.
Adenylate cyclase activity is conventionally assayed indirectly by measuring the synthesis of radioactively labeled cAMP from the substrate .alpha.-.sup.32 P-labeled ATP as described by Y. Salomon et at., as disclosed in Anal. Biochem., 58, 541 (1974) and Adv. Cyclic Nucleotide Res., 10, 35 (1979). The methods employ sequential affinity chromatography with Dowex exchange resin and aluminum oxide columns to separate newly generated [.sup.32 P]cAMP from [.alpha.-.sup.32 P]ATP. See also, C. L. Johnson et al., Mol. Pharmacol., 16, 417 (1979). Although this method is sensitive, it relies upon costly radioactively labeled compounds.
Previously, Lowry et al. have developed a number of sensitive assays which can measure small amounts of biological compounds based on the fluorescence of reduced pyridine nucleotides. See O. H. Lowry et al., A Flexible System of Enzymatic Analysis, Harcourt Brace Jovanovich, N.Y. (1972); F. M. Matschinsky et al., J. Histochem. Cytochem., 16, 29 (1968). These methods employ one or more of a series of enzymatic reactions which ultimately lead to the production of either .beta.-nicotinamide-adenine dinucleotide phosphate (NADP.sup.+) or .beta.-nicotinamide-adenine dinucleotide (NAD+) or the reduced forms NADPH and NADH. The reduced purine nucleotides can be precisely measured at O.D. 340 nm in a photometer. One reaction, the measurement of AMP, depends upon the stimulatory effects of AMP on glycogen phosphorylase a, the enzyme that converts glycogen into glucose-6-phosphate in the presence of inorganic phosphate (P.sub.i). See, E. Helmreich et al., Biochemistry, 52, 647 (1964); ibid., 51, 131 (1964); O. H. Lowry et al., J. Biol. Chem., 239, 1947 (1964); M. Trus et al., Diabetes, 29, 1 (1980). Attempts to increase the analytical sensitivity and specificity for AMP or AC have involved the enzymic degradation of interfering nucleotides and/or their removal by chromatography (See N. D. Goldberg et al., Anal Biochem, 28, 523 (1969); B. McL. Breckenridge, PNAS USA, 52, 1580 (1964)). However, fluorometric assays have not been developed which can either directly or indirectly measure adenylate cyclase activity in physiological samples needed to accurately quantify AC or cAMP in .mu.g samples at pmol or fmol levels.
Given the safety and environmental concerns associated with the use and disposal of radioactive materials used in the current methods to measure AC, a need exists for a highly sensitive nonradioactive assay to measure adenylate cyclase activity.